Therapeutic molecules for modulating stability of vegf
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example 1
Regulation of VEGF Expression
[0136]Oligonucleotides S1, S2 S3 and DS-085 (see Materials and Methods) were transfected into the RPE 51 cell line and the effects of VEGF translation and transcription were measured using ELISA and RT-PCR respectively. ELISA (FIG. 1) revealed that both S1 (1073 pg mL−1) and S2 (969 pg ml−1) facilitated a significant (p−1), oligonucleotide S3 (593 pg mL−1) had no significant effect (P>0.05) transfection agent cytofectin mediated a slight decrease in VEGF expression (508 pg mL−1). However, this result was not found to be significant (p>0.05). Regulation of vascular endothelial growth factor.
[0137]To examine the effects on VEGF at the transcriptional level, total RNA was extracted from cells transfected with oligonucleotide S1, S2, S3 and DS-085 and subsequently used as a template for RT-PCR (FIG. 2a). The profile for mRNA levels in the transfected cells as determined by densitometry of the PCR products (FIG. 2b) reflected the protein concentration profile...
example 2
In Vivo Analysis
[0143]To determine if the in vitro observations would translate into an in vivo effect, the oligonucleotides were injected into the anterior chamber of rat eyes. Subsequent opthalmologic examination showed strong neovascularization in the iris of rat eyes 7 days following injection with S1 and S2, (FIG. 3) but no effect was observed in rat eyes injected with S3 or vehicle. This indicates that the oligonucleotides were able to mediate the up-regulation of VEGF the eye and produce an angiogenic response.
[0144]Similarly, when the rats were injected in the sub-retinal space with S1 and S2 a strong angiogenic response was observed when viewed using color fundus photography. Neovascularization occurred some distance from the injection site and appeared as a distinct band extending across the retina (FIG. 4a). Further examination using fluorescein angiography confirmed the formation of new vessels which appears as hyperfluorescence (FIG. 4b) due to the “leaky” nature blood ...
example 3
[0147]Cross species comparisons of VEGF 5′-UTR sequences between bovine, murine and human has revealed a high level of conservation for the S1 to S2 region between human and bovine but the murine 5′-UTR revealed a complete lack of the S1 sequence (FIG. 5a). No sequence information is available for the 5′-UTR of the rat therefore no direct comparison can be made. Further examination of both the 5′- and 3′-UTR of the human VEGF gene has revealed several possible sites for destabilizing elements (FIG. 5b).
Discussion of the Examples
[0148]Controlled regulation of VEGF in vivo is important in maintaining the health of many tissues and cells types. However, increased levels of VEGF associated with ischemic conditions leads to a variety of angiogenic ocular diseases including diabetic retinopathy and retinopathy of prematurity (23,24), in addition to promoting vasculogenesis in cancerous tissues (25,26). Central to the regulation of VEGF is the presence of both a 5′-UTR, ...
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